The ability to transfer plasmids into E. coli has come to be an integral part of the repertoire of tools used in molecular biology. Plasmid transformation of E. coli was first observed by Cohen, et al, Proc. Natl. Acad. Sci. 69: 2110 (1972), by applying the observation of Mandel, et al, J. Mol. Biol. 53: 159 (1970) that E. coli and bacteriophage .lambda., when combined in CaCl.sub.2 containing solutions at 0.degree. C. caused transfection of the E. coli with the phage.
Much work has been undertaken since then, with the goals of improving transformation frequency, and characterizing the parameters involved. Examples of this work include Hanahan, J. Mol. Biol. 166: 557-580 (1983), which is incorporated by reference herein; Bergmans, et al, J. Bacteriol 146: 564 (1981); Jones, et al, J. Bacteriol 146: 841 (1981); Norgard, et al, Gene 3: 279 (1978).
What the art as a whole teaches, in summary, is that E. coli and DNA interact at low temperatures in an environment containing divalent cations. Many factors improve transformation frequency, including heat shock, inclusion of monovalent cations in the transforming buffer, the addition of hexamine cobalt chloride, treatment with solvents and sulfhydryl reagents, and growth in media containing elevated magnesium levels. These treatments have improved transformation from 1 plasmid in 10.sup.5 cells, to up to 1 in 10.sup.2 cells.
One cell line which has been of particular use in this regard is cell line DH-1, which is described in Hanahan, J. Mol. Biol. 166: 557-580 (1983). This cell line is an E. coli mutant (recAI), and has served as a host for, e.g., cDNA cloning, plasmid rescue, and cloning of large DNA fragments.
Recently, it has been learned that a mutation, identified as deoR, can be induced in E. coli cells. When present, the cells which carry the mutant serve as unexpectedly better hosts for all of the techniques described supra, as compared to DH-1. The DH-1 cell line itself has been mutated to contain the deoR mutation, and the resulting cell line is known as DH-5.
Cell line DH-5 has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville Maryland and bears accession number 53868. This deposit affords permanency of cell line and provides ready access to the public.
A method for obtaining deoR mutated E. coli cell lines, the mutated cell lines themselves, and methods of using these for, e.g., cell transformation, are the subject of this invention.
While the deoR mutations might occur in nature, substantially pure cultures of these have never before been available. The inventions described herein provides a method to one skilled in the art whereby a regular source of the mutant line now becomes available.